Discovery of selective, small-molecule inhibitors of RNA complexes--I. The Tat protein/TAR RNA complexes required for HIV-1 transcription

We have developed a therapeutic program focusing on the inhibition of a human immunodeficiency virus-1 specific protein-RNA interaction. This program begins with a search for small organic molecules that would interfere with the binding of Tat protein to TAR RNA. The methodologies chosen to study the HIV-1 Tat-TAR interaction and inhibition include gel mobility shift assays, scintillation proximity assays, filtration assays, and mass spectrometry. These methods helped establish in vitro high-throughput screening assays which rapidly identified Tat-TAR inhibitors from our corporate compound library. Tat-activated reporter gene assays were then used to investigate the cellular activities of the Tat-TAR inhibitors. The cellular activity, selectivity, and toxicity data for select Tat-TAR inhibitors were determined. Evaluation of both the cellular data and the Tat-TAR inhibition results led to further testing in anti-HIV-1 infection assays.     

Dietary variety via sweetening and voluntary feed intake of lactating dairy cows

The effects of choice of diets on feed intake were studied using 12 lactating Holstein cows. A switchback design was used that had three periods and two sequential blocks. Diets were 1) a control total mixed ration (TMR), which consisted of alfalfa haylage, corn silage, and a concentrate mixture based on ground corn and soybean meal (25:25:50 on a dry matter basis) and 2) a sweetened TMR in which a brown sugar food product constituted 1.5% of the dietary dry matter. Treatments consisted of the control TMR fed on both sides of divided feed bunks, the sweetened TMR fed on both sides of divided feed bunks, or both TMR fed on alternating (daily) sides of divided feed bunks in tie stalls. Periods were 2 wk in duration, and cows averaged 67 and 53 d of lactation at the start of blocks 1 and 2, respectively. The dry matter intake, body weight, milk yield, and percentages of milk fat, protein, and solids not fat were similar when either TMR was fed alone. A choice of control TMR or sweetened TMR did not affect any of these variables. The dry matter intake, body weight, milk yield, and milk protein percentage were affected by block; however, these effects were probably caused by differences between the blocks in environment and stage of lactation. The results of this experiment might have been affected by the composition of the control TMR, its similarity to the sweetened TMR, availability of both diets simultaneously when a choice was offered, and use of a TMR instead of separate feeds or simpler mixtures.     

Nitrogen mustard up-regulates Bcl-2 and GSH and increases NTP and PCr in HT-29 colon cancer cells

We hypothesized that unexplained increases in nucleoside triphosphates (NTP) observed by 31P magnetic resonance spectroscopy (MRS) after treatment of tumours by DNA-damaging agents were related to chemotherapy-induced up-regulation of the bcl-2 gene and DNA damage prevention and repair processes. To test this hypothesis, we treated HT-29 cells with 10(-4) M nitrogen mustard (HN2) and performed sequential perchloric acid extractions in replicate over 0-18 h. By reference to an internal standard (methylene diphosphonic acid), absolute changes in 31P-detectable high-energy phosphates in these extracts were determined and correlated with changes in bcl-2 protein levels, cell viability, cell cycle, apoptosis and total cellular glutathione (GSH) (an important defence against DNA damage from alkylating agents). After HN2 administration, bcl-2 protein levels in the HT-29 cell line rose at 2 h. Cell viability declined to 25% within 18 h, but apoptosis measured using fluorescence techniques remained in the 1-4% range. Increased cell division was noted at 4 h. Two high-energy interconvertible phosphates, NTP (P < or = 0.006) and phosphocreatine (PCr) (P < or = 0.0002), increased at 2 h concurrently with increased levels of bcl-2 protein and glutathione. This study demonstrates that bcl-2 and glutathione are up-regulated by HN2 and links this to a previously unexplained 31P MRS phenomenon: increased NTP after chemotherapy.     

An examination of the distinction between nouns and verbs: associations with two different kinds of motion

Four experiments provide evidence that people are biased to associate particular types of motion with nouns and different types of motion with verbs. Novel nouns and verbs were related to two types of motion: (1) path, or the direction of motion of one character with respect to the other character, and (2) movement orientation, or the direction a character was facing as it moved. Subjects associated verbs more strongly with path than with movement orientation. In contrast, they associated nouns more strongly with movement orientation than with path. Movement orientation was associated with both object categories and verbs, inconsistent with a complete division of labor between these two types of categories. These results are consistent, however, with the notion that people are biased to associate verbs with relations between objects, whereas they are biased to associate object categories with motions defined with respect to the object carrying out those motions.